Methods of using FET labeled oligonucleotides that include a 3&#39;-5&#39; exonuclease resistant quencher domain and compositions for practicing the same

ABSTRACT

Methods and compositions are provided for detecting a primer extension product in a reaction mixture. In the subject methods, a primer extension reaction is conducted in the presence of a polymerase having 3′→5′ exonuclease activity and at least one FET labeled oligonucleotide probe that includes a 3′→5′ exonuclease resistant quencher domain. Also provided are systems and kits for practicing the subject methods. The subject invention finds use in a variety of different applications, and are particularly suited for use in high fidelity PCR based reactions, including SNP detection applications, allelic variation detection applications, and the like.

INTRODUCTION

[0001] 1. Technical Field

[0002] The technical field of this invention is the polymerase chainreaction (PCR); and particularly high fidelity PCR.

[0003] 2. Background of the Invention

[0004] The polymerase chain reaction (PCR) is a powerful method for therapid and exponential amplification of target nucleic acid sequences.PCR has facilitated the development of gene characterization andmolecular cloning technologies including the direct sequencing of PCRamplified DNA, the determination of allelic variation, and the detectionof infectious and genetic disease disorders. PCR is performed byrepeated cycles of heat denaturation of a DNA template containing thetarget sequence, annealing of opposing primers to the complementary DNAstrands, and extension of the annealed primers with a DNA polymerase.Multiple PCR cycles result in the exponential amplification of thenucleotide sequence delineated by the flanking amplification primers.

[0005] An important modification of the original PCR technique was thesubstitution of Thermus aquaticus (Taq) DNA polymerase in place of theKlenow fragment of E. coli DNA pol I (Saiki, et al. Science,230:1350-1354 (1988)). The incorporation of a thermostable DNApolymerase into the PCR protocol obviates the need for repeated enzymeadditions and permits elevated annealing and primer extensiontemperatures which enhance the specificity of primer:templateassociations. Taq DNA polymerase thus serves to increase the specificityand simplicity of PCR.

[0006] Although Taq DNA polymerase is used in the vast majority of PCRperformed today, it has a fundamental drawback: purified Taq DNApolymerase enzyme is devoid of 3′ to 5′ exonuclease activity and thuscannot excise misinserted nucleotides (Tindall, et al., Biochemistry,29:5226-5231 (1990)). Consistent with these findings, the observed errorrate (mutations per nucleotide per cycle) of Taq polymerase isrelatively high; estimates range from 2×10⁻⁴ during PCR (Saiki et al.,Science, 239:487-491 (1988); Keohavaong et al. Proc. Natl. Acad. Sci.USA, 86:9253-9257 (1989)) to 2×10⁻⁵ for base substitution errorsproduced during a single round of DNA synthesis of the lacZ gene (Eckertet al., Nucl. Acids Res. 18:3739-3744 (1990)).

[0007] Polymerase induced mutations incurred during PCR increasearithmetically as a function of cycle number. For example, if an averageof two mutations occur during one cycle of amplification, 20 mutationswill occur after 10 cycles and 40 will occur after 20 cycles. Eachmutant and wild type template DNA molecule will be amplifiedexponentially during PCR and thus a large percentage of the resultingamplification products will contain mutations. Mutations introduced byTaq DNA polymerase during DNA amplification have hindered PCRapplications that require high fidelity DNA synthesis. Severalindependent studies suggest that 3′ to 5′ exonuclease-dependentproofreading enhances the fidelity of DNA synthesis (Reyland et al, J.Biol. Chem., 263:6518-6524, 1988; Kunkel et al, J. Biol. Chem.,261:13610-13616, 1986; Bernad et al, Cell, 58:219-228, 1989). As such,it is desirable, where possible, to include a 3′ to 5′exonuclease-dependent proofreading activity in PCR based reactions. Forexample, If Taq DNA Polymerase (error rate 2×10⁻⁴) is used to amplify a100 bp sequence for 40 cycles by PCR, about 55% of the amplificationproducts will contain one or more errors. In contrast, if a Pwo DNAPolymerase having proof-reading activities is used for theamplification, only 10% of the products will contain an error under thesame conditions. The error rate produced by a mixture of Taq DNAPolymerase and a proofreading DNA Polymerase between these two values(Cline et al, Nucleic Acids Res., 24(18):3546-51, 1996).

[0008] In many PCR based reactions, a signal producing system isemployed, e.g., to detect the production of amplified product. One typeof signal producing system that is attractive for use in PCR basedreactions is the fluorescence energy transfer (FET) system, in which anucleic acid detector includes fluorescence donor and acceptor groups.FET label systems include a number of advantages over other labelingsystems, including the ability to perform homogeneous assays in which aseparation step of bound vs. unbound labeled nucleic acid detector isnot required.

[0009] In such real time detection systems using a FET labeled nucleicacid detector, high fidelity amplification is critical. Any error insequences where a FET labeled nucleic acid detector binds can causeprobes not to bind or wrong probes to bind in the case of allelediscrimination, resulting in weak signal or the wrong signal beingproduced. For example, if a 30 bp PCR fragment which is the target of aFET labeled probe is amplified using Taq DNA Polymerase for 40 cycles,about 22% of the amplification fragments will contain one or moreerrors. In contrast, if a Pwo DNA Polymerase having proof-readingactivities is used for the amplification, only 3% of the amplificationfragments will contain an error under the same conditions. Therefore,the standard low fidelity amplification can cause a decrease insensitivity or mis-typing in the case of allele discrimination.

[0010] However, as discovered by the current invention a disadvantage ofcurrently available FET labeled nucleic acids having TAMRA or Dabcyl asa quencher is that such nucleic acids are subject to 3′→5′ exonucleasedegradation. Accordingly, such FET labeled nucleic acids are notsuitable for use in high fidelity PCR applications, where 3′→5′exonuclease activity, i.e., proofreading activity, is present.

[0011] As such, there is significant interest in the identification anddevelopment of FET labeled nucleic acids that can be used in highfidelity PCR applications.

[0012] Relevant Literature

[0013] U.S. patents of interest include: U.S. Pat. Nos. 5,538,848 and6,248,526. Also of interest are: WO 01/86001 and WO 01/42505.

SUMMARY OF THE INVENTION

[0014] Methods and compositions are provided for detecting a primerextension product in a reaction mixture. In the subject methods, aprimer extension reaction is conducted in the presence of a polymerasehaving 3′→5′ exonuclease activity and at least one FET labeledoligonucleotide probe that includes a 3′→5′ exonuclease resistantquencher domain. Also provided are systems and kits for practicing thesubject methods. The subject invention finds use in a variety ofdifferent applications, and is particularly suited for use in highfidelity PCR based reactions, including SNP detection applications,allelic variation detection applications, and the like.

[0015] Definitions

[0016] As used herein, “nucleic acid” means either DNA, RNA,single-stranded or double-stranded, and any chemical modificationsthereof. Modifications include, but are not limited to, those whichprovide other chemical groups that incorporate additional charge,polarizability, hydrogen bonding, electrostatic interaction, andfunctionality to the nucleic acid. Such modifications include, but arenot limited to, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil; backbone modifications, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine and the like. Modifications can also include 3′ and 5′modifications such as capping.

[0017] As used herein, “fluorescent group” refers to a molecule that,when excited with light having a selected wavelength, emits light of adifferent wavelength. Fluorescent groups may also be referred to as“fluorophores”.

[0018] As used herein, “fluorescence-modifying group” refers to amolecule that can alter in any way the fluorescence emission from afluorescent group. A fluorescence-modifying group generally accomplishesthis through an energy transfer mechanism. Depending on the identity ofthe fluorescence-modifying group, the fluorescence emission can undergoa number of alterations, including, but not limited to, attenuation,complete quenching, enhancement, a shift in wavelength, a shift inpolarity, a change in fluorescence lifetime. One example of afluorescence-modifying group is a quenching group.

[0019] As used herein, “energy transfer” refers to the process by whichthe fluorescence emission of a fluorescent group is altered by afluorescence-modifying group. If the fluorescence-modifying group is aquenching group, then the fluorescence emission from the fluorescentgroup is attenuated (quenched). Energy transfer can occur throughfluorescence resonance energy transfer, or through direct energytransfer. The exact energy transfer mechanisms in these two cases aredifferent. It is to be understood that any reference to energy transferin the instant application encompasses all of thesemechanistically-distinct phenomena. Energy transfer is also referred toherein as fluorescent energy transfer or FET.

[0020] As used herein, “energy transfer pair” refers to any twomolecules that participate in energy transfer. Typically, one of themolecules acts as a fluorescent group, and the other acts as afluorescence-modifying group. The preferred energy transfer pair of theinstant invention comprises a fluorescent group and a quenching group.In some cases, the distinction between the fluorescent group and thefluorescence-modifying group may be blurred. For example, under certaincircumstances, two adjacent fluorescein groups can quench one another'sfluorescence emission via direct energy transfer. For this reason, thereis no limitation on the identity of the individual members of the energytransfer pair in this application. All that is required is that thespectroscopic properties of the energy transfer pair as a whole changein some measurable way if the distance between the individual members isaltered by some critical amount.

[0021] “Energy transfer pair” is used to refer to a group of moleculesthat form a single complex within which energy transfer occurs. Suchcomplexes may comprise, for example, two fluorescent groups which may bedifferent from one another and one quenching group, two quenching groupsand one fluorescent group, or multiple fluorescent groups and multiplequenching groups. In cases where there are multiple fluorescent groupsand/or multiple quenching groups, the individual groups may be differentfrom one another.

[0022] As used herein, “quenching group” refers to anyfluorescence-modifying group that can attenuate at least partly thelight emitted by a fluorescent group. We refer herein to thisattenuation as “quenching”. Hence, illumination of the fluorescent groupin the presence of the quenching group leads to an emission signal thatis less intense than expected, or even completely absent. Quenchingoccurs through energy transfer between the fluorescent group and thequenching group.

[0023] As used herein, “fluorescence resonance energy transfer” or“FRET” refers to an energy transfer phenomenon in which the lightemitted by the excited fluorescent group is absorbed at least partiallyby a fluorescence-modifying group. If the fluorescence-modifying groupis a quenching group, then that group can either radiate the absorbedlight as light of a different wavelength, or it can dissipate it asheat. FRET depends on an overlap between the emission spectrum of thefluorescent group and the absorption spectrum of the quenching group.FRET also depends on the distance between the quenching group and thefluorescent group. Above a certain critical distance, the quenchinggroup is unable to absorb the light emitted by the fluorescent group, orcan do so only poorly.

[0024] As used herein “direct energy transfer” refers to an energytransfer mechanism in which passage of a photon between the fluorescentgroup and the fluorescence-modifying group does not occur. Without beingbound by a single mechanism, it is believed that in direct energytransfer, the fluorescent group and the fluorescence-modifying groupinterfere with each others electronic structure. If thefluorescence-modifying group is a quenching group, this will result inthe quenching group preventing the fluorescent group from even emittinglight.

[0025] In general, quenching by direct energy transfer is more efficientthan quenching by FRET. Indeed, some quenching groups that do not quenchparticular fluorescent groups by FRET (because they do not have thenecessary spectral overlap with the fluorescent group) can do soefficiently by direct energy transfer. Furthermore, some fluorescentgroups can act as quenching groups themselves if they are close enoughto other fluorescent groups to cause direct energy transfer. Forexample, under these conditions, two adjacent fluorescein groups canquench one another's fluorescence effectively. For these reasons, thereis no limitation on the nature of the fluorescent groups and quenchinggroups useful for the practice of this invention.

[0026] An example of “stringent hybridization conditions” ishybridization at 50° C. or higher and 6.0×SSC (900 mM NaCl/90 mM sodiumcitrate). Another example of stringent hybridization conditions isovernight incubation at 42° C. or higher in a solution: 50% formamide,6×SSC (900 mM NaCl, 90 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon spermDNA. Stringent hybridization conditions are hybridization conditionsthat are at least as stringent as the above representative conditions,where conditions are considered to be at least as stringent if they areat least about 80% as stringent, typically at least about 90% asstringent as the above specific stringent conditions. Other stringenthybridization conditions are known in the art and may also be employed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIGS. 1A to 1E provide graphical results of assays comparing thefunction of various TET labeled FET probes under high fidelity andstandard PCR conditions.

[0028]FIGS. 2A to 2B provide graphical results of assays comparing thefunction of FAM/BHQ1 FET probe and FAM/TAMRA FET probe under highfidelity and standard PCR conditions.

[0029]FIG. 3 provides graphical results of a multicomponent analysis ofa FAM/TAMRA FET oligo primer and a FAM/BHQ1 FET oligo primer.

[0030]FIG. 4 provides graphical results of assays for allelediscrimination using FAM and TET labeled FET probes under high fidelityPCR conditions.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0031] Methods and compositions are provided for detecting a primerextension product in a reaction mixture. In the subject methods, aprimer extension reaction is conducted in the presence of a polymerasehaving 3′→5′ exonuclease activity and at least one FET labeledoligonucleotide probe that includes a 3′→5′ exonuclease resistantquencher domain. Also provided are systems and kits for practicing thesubject methods. The subject invention finds use in a variety ofdifferent applications, and is particularly suited for use in highfidelity PCR based reactions, including SNP detection applications,allelic variation detection applications, and the like.

[0032] Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

[0033] In this specification and the appended claims, the singular forms“a,” “an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

[0034] Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

[0035] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0036] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing those componentsthat are described in the publications which might be used in connectionwith the presently described invention.

[0037] As summarized above, the subject invention provides methods ofdetecting the production of a primer extension product in a primerextension reaction mixture by using a FET labeled oligonucleotide probe.In further describing the subject invention, the methods are describedfirst in greater detail, followed by a review of representative specificapplications in which the methods finds use, as well as systems and kitsthat find use in practicing the subject methods.

[0038] Methods

[0039] As summarized above, the subject invention provides methods fordetecting the production of primer extension products in a primerextension reaction mixture. In other words, the subject inventionprovides methods of determining whether primer extension products areproduced in a primer extension reaction. By primer extension product ismeant a nucleic acid molecule that results from a template dependentprimer extension reaction. Template dependent primer extension reactionsare those reactions in which a polymerase extends a nucleic acid primermolecule that is hybridized to a template nucleic acid molecule, wherethe sequence of bases that is added to the terminus of the primernucleic acid molecule is determined by the sequence of bases in thetemplate strand. Template dependent primer extension reactions includeboth amplification and non-amplification primer extension reactions. Inmany embodiments of the subject invention, the template dependent primerextension reaction in which the production of primer extension productsis detected is an amplification reaction, e.g., a polymerase chainreaction (PCR).

[0040] A feature of the subject methods is that the template dependentprimer extension reaction in which the production of primer extensionproducts is detected is a “high fidelity” reaction. By “high fidelity”reaction is meant that the reaction has a low error rate, i.e., a lowrate of wrong nucleotide incorporation. As such, the error rate of thesubject reactions is typically less than about 2×10⁻⁴, usually less thanabout 1×10⁻⁵ and more usually less than about 1×10⁻⁶.

[0041] Preparation of Reaction Mixture

[0042] In practicing the subject methods, the first step is to produce a“high fidelity” primer extension mixture, e.g., a composition of matterthat includes all of the elements necessary for a high fidelity primerextension reaction to occur, where the primer extension mixture furtherincludes at least one FET labeled oligonucleotide that includes a 3′→5′exonuclease resistant quencher domain.

[0043] FET occurs when a suitable fluorescent energy donor and an energyacceptor moiety are in close proximity to one another. The excitationenergy absorbed by the donor is transferred to the acceptor which canthen further dissipate this energy either by fluorescent emission if afluorophore, or by non-fluorescent means if a quencher. A donor-acceptorpair comprises two fluorophores having overlapping spectra, where thedonor emission overlaps the acceptor absorption, so that there is energytransfer from the excited fluorophore to the other member of the pair.It is not essential that the excited fluorophore actually fluoresce, itbeing sufficient that the excited fluorophore be able to efficientlyabsorb the excitation energy and efficiently transfer it to the emittingfluorophore.

[0044] As such, the FET labeled oligonucleotides employed in the subjectmethods are nucleic acid detectors that include a fluorophore domainwhere the fluorescent energy donor, i.e., donor, is positioned and anacceptor domain where the fluorescent energy acceptor, i.e., acceptor,is positioned. As mentioned above, the donor domain includes the donorfluorophore. The donor fluorophore may be positioned anywhere in thenucleic acid detector, but is typically present at the 5′ terminus ofthe detector.

[0045] The acceptor domain includes the fluorescence energy acceptor.The acceptor may be positioned anywhere in the acceptor domain, but istypically present at the 3′ terminus of the nucleic acid detector.

[0046] In addition to the fluorophore and acceptor domains, the FETlabeled oligonucleotides also include a target nucleic acid bindingdomain, which binds to a target nucleic acid sequence, e.g., understringent hybridization conditions (as defined above). This targetbinding domain typically ranges in length from about 10 to about 60 nt,usually from about 15 to about 30 nt. Depending on the nature of theoligonucleotide and the assay itself, the target binding domain mayhybridize to a region of the template nucleic acid or a region of theprimer extension product. For example, where the assay is a 5′ nucleaseassay, e.g., in which a Taqman type oligonucleotide probe is employed,the target binding domain hybridizes under stringent conditions to atarget binding site of the template nucleic acid, which is downstream or3′ of the primer binding site. In alternative embodiments, e.g., inmolecular beacon type assays, the target binding domain hybridizes to adomain of a primer extension product.

[0047] The overall length of the FET labeled oligonucleotides, whichincludes all three domains mentioned above, typically ranges from about10 to about 60 nt, usually from about 15 to about 30 nt.

[0048] The donor fluorophore of the subject probes is typically one thatis excited efficiently by a single light source of narrow bandwidth,particularly a laser source. The emitting or accepting fluorophores areselected to be able to receive the energy from the donor fluorophore andemit light. Usually the donor fluorophores will absorb in the range ofabout 350-800 nm, more usually in the range of about 350-600 nm or500-750 nm. The transfer of the optical excitation from the donor to theacceptor depends on the distance between the two fluorophores. Thus, thedistance must be chosen to provide efficient energy transfer from thedonor to the acceptor. The distance between the donor and acceptormoieties on the FET oligonucleotides employed in the subject invention,at least in certain configurations (such as upon intramolecularassociation) typically ranges from about 10 to about 100 angstroms

[0049] The fluorophores for FET pairs may be selected so as to be from asimilar chemical family or a different one, such as cyanine dyes,xanthenes or the like. Fluorophores of interest include, but are notlimited to: fluorescein dyes (e.g., 5-carboxyfluorescein (5-FAM),6-carboxyfluorescein (6-FAM), 2′,4′,1,4,-tetrachlorofluorescein (TET),2′,4′,5′,7′,1,4-hexachlorofluorescein (HEX), and2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE)), cyanine dyessuch as Cy5, dansyl derivatives, rhodamine dyes (e.g.,tetramethyl-6-carboxyrhodamine (TAMRA), andtetrapropano-6-carboxyrhodamine (ROX)), DABSYL, DABCYL, cyanine, such asCy3, anthraquinone, nitrothiazole, and nitroimidazole compounds, and thelike. Fluorophores of interest are further described in WO 01/42505 andWO 01/86001, as well as the priority U.S. applications of thesedocuments, the disclosures of the latter of which are hereinincorporated by reference.

[0050] A feature of the subject FET labeled oligonucleotides is thatthey are 3′→5′ exonuclease resistant. As such, they are not degraded by3′→5′ exonucleases, i.e., enzymes having 3′→5′ exonuclease activity. The3′→5′ exonuclease resistance of the subject FET labeled oligonucleotidesmay arise from the presence of the acceptor moiety present in theacceptor domain of the FET labeled oligonucleotide. In many, though notall embodiments, the acceptor moiety is present at the 3′ terminus ofthe acceptor domain, and in many embodiments at the 3′ terminus of theFET labeled oligonucleotide as a whole.

[0051] Any acceptor or donor that imparts 3′→5′ exonuclease resistanceonto the FET labeled oligonucleotides may be employed. In manyembodiments, the acceptor moiety is a quencher molecule, e.g., amolecule that absorbs transferred energy but does not emit fluorescence,e.g., a dark quencher. In many embodiments, the dark quencher hasmaximum absorbance of between about 400 and about 700 nm, and oftenbetween about 500 and about 600 nm.

[0052] In certain embodiments, the dark quencher comprises a substituted4-(phenyidiazenyl)phenylamine structure, often comprising at least tworesidues selected from aryl, substituted aryl, heteroaryl, substitutedheteroaryl and combination thereof, wherein at least two of saidresidues are covalently linked via an exocyclic diazo bond.

[0053] In certain embodiments, the dark quencher is described by thefollowing formula:

[0054] wherein:

[0055] R₀,R₁,R₂,R₃,R₄,R₅ are independently: —H, halogen, —O(CH₂)_(n)CH₃,—(CH₂)_(n)CH₃, —NO₂, SO₃, —N[(CH₂)_(n)CH₃]₂ wherein n=0 to 5 or —CN;

[0056] R₆ is —H or —(CH₂)_(n)CH₃ where n=0 to 5; and

[0057] v is a number from 0 to 10.

[0058] Dark quenchers of interest are further described in WO 01/42505and WO 01/86001, as well as the priority U.S. applications of thesedocuments, the disclosures of the latter of which are hereinincorporated by reference.

[0059] The FET labeled oligonucleotide may be structured in a variety ofdifferent ways, so long as it includes the above described donor,acceptor and target nucleic acid binding domains. Typically, the FETlabeled oligonucleotide is structured such that energy transfer occursbetween the fluorophore and acceptor of the FET labeled oligonucleotideprobe upon fluorophore excitation when the FET labeled oligonucleotideis not hybridized to target nucleic acid.

[0060] In certain embodiments, the oligonucleotide is a single strandedmolecule that does not form intramolecular structures and in whichenergy transfer occurs because the spacing of the donor and acceptorprovides for energy transfer in the single stranded linear format. Inthese embodiments, energy transfer also occurs between the fluorophoreand acceptor of FET labeled oligonucleotide probe upon fluorophoreexcitation when the FET labeled oligonucleotide probe is hybridized to atarget nucleic acid. Specific examples of such FET labeledoligonucleotide probes include the Taqman™ type probes, as described inU.S. Pat. No. 6,248,526, the disclosure of which is herein incorporatedby reference (as well as Held et al., Genome Res. (1996) 6:986-994;Holland et al., Proc. Nat'l Acad. Sci. USA (1991) 88:7276-7280; and Leeet al., Nuc. Acids Res. (1993) 21:3761-3766 (1993)). In many of theseembodiments, the target nucleic acid binding domain is one thathybridizes to, i.e., is complementary to, a sequence of the templatenucleic acid, i.e., the target nucleic acid of the target nucleic acidbinding domain is a sequence present in the template nucleic acid.

[0061] In other embodiments, the probe oligonucleotides are structuredsuch that energy transfer does not occur between the fluorophore andacceptor of said FET labeled oligonucleotide probe upon fluorophoreexcitation when the FET labeled oligonucleotide probe is hybridized to atarget nucleic acid. Examples of these types of probe structuresinclude: Scorpion probes (as described in Whitcombe et al., (NatureBiotechnology (1999) 17:804-807; U.S. Pat. No. 6,326,145, the disclosureof which is herein incorporated by reference), Sunrise probes (asdescribed in Nazarenko et al., Nuc. Acids Res. (1997) 25:2516-2521; U.S.Pat. No. 6,117,635, the disclosure of which is herein incorporated byreference), Molecular Beacons (Tyagi et al., Nature Biotechnology (1996)14:303-308; U.S. Pat. No. 5,989,823, the disclosure of which isincorporated herein by reference), and conformationally assisted probes(as described in provisional application serial no. 60/138,376, thedisclosure of which is herein incorporated by reference). In many ofthese embodiments, the target binding sequence or domain comprises ahybridization domain complementary to a sequence of the primer extensionproduct.

[0062] Since the primer extension reaction mixture produced in theinitial step of the subject methods is a high fidelity primer extensionreaction mixture, it further includes an enzyme having 3′→5′ exonucleaseactivity. In many embodiments, the 3′→5′ exonuclease is a polymerasethat has 3′→5′ exonuclease activity. In many embodiments, the highfidelity nature of the reaction mixture is provided by the presence of acombination of two or more polymerases, at least one of which includes a3′→5′ exonuclease. In certain embodiments, e.g., in 5′ nucleaseapplications, care is taken to ensure that a polymerase having 5′→3′nuclease activity is also included. In many embodiments, the polymerasecombination employed includes at least one Family A polymerase and, inmany embodiments, a Family A polymerase and a Family B polymerase, wherethe terms “Family A” and “Family B” correspond to the classificationscheme reported in Braithwaite & Ito, Nucleic Acids Res. (1993)21:787-802. Family A polymerases of interest include: Thermus aquaticuspolymerases, including the naturally occurring polymerase (Taq) andderivatives and homologues thereof, such as Klentaq (as described inProc. Natl. Acad. Sci USA (1994) 91:2216-2220); Thermus thermophiluspolymerases, including the naturally occurring polymerase (Tth) andderivatives and homologues thereof, and the like. Family B polymerasesof interest include Thermococcus litoralis DNA polymerase (Vent) asdescribed in Perler et al., Proc. Natl. Acad. Sci. USA (1992) 89:5577;Pyrococcus species GB-D (Deep Vent); Pyrococcus furiosus DNA polymerase(Pfu) as described in Lundberg et al., Gene (1991) 108:1-6, Pyrococcuswoesei (Pwo) and the like. Of the two types of polymerases employed, theFamily A polymerase will typically be present the reaction mixture in anamount greater than the Family B polymerase, where the difference inactivity will usually be at least 10-fold, and more usually at leastabout 100-fold. Accordingly, the reaction mixture will typicallycomprise from about 0.1 U/I to 1 U/I Family A polymerase, usually fromabout 0.2 to 0.5 U/I Family A polymerase, while the amount of Family Bpolymerase will typically range from about 0.01 mU/I to 10 mU/I, usuallyfrom about 0.05 to 1 mU/I and more usually from about 0.1 to 0.5 mU/I,where “U” corresponds to incorporation of 10 nmol dNTP intoacid-insoluble material in 30 min at 74° C.

[0063] Another component of the reaction mixture produced in the firststep of the subject methods is the template nucleic acid. The nucleicacid that serves as template may be single stranded or double stranded,where the nucleic acid is typically deoxyribonucleic acid (DNA). Thelength of the template nucleic acid may be as short as 50 bp, butusually be at least about 100 bp long, and more usually at least about150 bp long, and may be as long as 10,000 bp or longer, e.g., 50,000 bpin length or longer, including a genomic DNA extract, or digest thereof,etc. The nucleic acid may be free in solution, flanked at one or bothends with non-template nucleic acid, present in a vector, e.g. plasmidand the like, with the only criteria being that the nucleic acid beavailable for participation in the primer extension reaction. Thetemplate nucleic acid may be present in purified form, or in a complexmixture with other non-template nucleic acids, e.g., in cellular DNApreparation, etc.

[0064] The template nucleic acid may be derived from a variety ofdifferent sources, depending on the application for which the PCR isbeing performed, where such sources include organisms that comprisenucleic acids, i.e. viruses; prokaryotes, e.g. bacteria, archaea andcyanobacteria; and eukaryotes, e.g. members of the kingdom protista,such as flagellates, amoebas and their relatives, amoeboid parasites,ciliates and the like; members of the kingdom fungi, such as slimemolds, acellular slime molds, cellular slime molds, water molds, truemolds, conjugating fungi, sac fungi, club fungi, imperfect fungi and thelike; plants, such as algae, mosses, liverworts, hornworts, club mosses,horsetails, ferns, gymnosperms and flowering plants, both monocots anddicots; and animals, including sponges, members of the phylum cnidaria,e.g. jelly fish, corals and the like, combjellies, worms, rotifers,roundworms, annelids, molluscs, arthropods, echinoderms, acorn worms,and vertebrates, including reptiles, fishes, birds, snakes, and mammals,e.g. rodents, primates, including humans, and the like. The templatenucleic acid may be used directly from its naturally occurring sourceand/or preprocessed in a number of different ways, as is known in theart. In some embodiments, the template nucleic acid may be from asynthetic source.

[0065] The next component of the reaction mixture produced in the firststep of the subject methods is the primers employed in the primerextension reaction, e.g., the PCR primers (such as forward and reverseprimers employed in geometric amplification or a single primer employedin a linear amplification). The oligonucleotide primers with which thetemplate nucleic acid (hereinafter referred to as template DNA forconvenience) is contacted will be of sufficient length to provide forhybridization to complementary template DNA under annealing conditions(described in greater detail below) but will be of insufficient lengthto form stable hybrids with template DNA under polymerizationconditions. The primers will generally be at least 10 bp in length,usually at least 15 bp in length and more usually at least 16 bp inlength and may be as long as 30 bp in length or longer, where the lengthof the primers will generally range from 18 to 50 bp in length, usuallyfrom about 20 to 35 bp in length. The template DNA may be contacted witha single primer or a set of two primers (forward and reverse primers),depending on whether primer extension, linear or exponentialamplification of the template DNA is desired. Where a single primer isemployed, the primer will typically be complementary to one of the 3′ends of the template DNA and when two primers are employed, the primerswill typically be complementary to the two 3′ ends of the doublestranded template DNA.

[0066] In addition to the above components, the reaction mixtureproduced in the subject methods includes deoxyribonucleosidetriphosphates (dNTPs). Usually the reaction mixture will comprise fourdifferent types of dNTPs corresponding to the four naturally occurringbases are present, i.e. dATP, dTTP, dCTP and dGTP. In the subjectmethods, each dNTP will typically be present in an amount ranging fromabout 10 to 5000 M, usually from about 20 to 1000 M.

[0067] The reaction mixture prepared in the first step of the subjectmethods further includes an aqueous buffer medium which includes asource of monovalent ions, a source of divalent cations and a bufferingagent. Any convenient source of monovalent ions, such as KCl, K-acetate,NH₄-acetate, K-glutamate, NH₄Cl, ammonium sulfate, and the like may beemployed. The divalent cation may be magnesium, manganese, zinc and thelike, where the cation will typically be magnesium. Any convenientsource of magnesium cation may be employed, including MgCl₂, Mg-acetate,and the like. The amount of Mg²⁺ present in the buffer may range from0.5 to 10 mM, but will preferably range from about 3 to 6 mM, and willideally be at about 5 mM. Representative buffering agents or salts thatmay be present in the buffer include Tris, Tricine, HEPES, MOPS and thelike, where the amount of buffering agent will typically range fromabout 5 to 150 mM, usually from about 10 to 100 mM, and more usuallyfrom about 20 to 50 mM, where in certain preferred embodiments thebuffering agent will be present in an amount sufficient to provide a pHranging from about 6.0 to 9.5, where most preferred is pH 7.3 at 72° C.Other agents which may be present in the buffer medium include chelatingagents, such as EDTA, EGTA and the like.

[0068] In preparing the reaction mixture, the various constituentcomponents may be combined in any convenient order. For example, thebuffer may be combined with primer, polymerase and then template DNA, orall of the various constituent components may be combined at the sametime to produce the reaction mixture.

[0069] Subjecting the Primer Extension Mixture to Primer ExtensionReaction Conditions

[0070] Following preparation of the reaction mixture, the reactionmixture is subjected to primer extension reaction conditions, i.e., toconditions that permit for polymerase mediated primer extension byaddition of nucleotides to the end of the primer molecule using thetemplate strand as a template. In many embodiments, the primer extensionreaction conditions are amplification conditions, which conditionsinclude a plurality of reaction cycles, where each reaction cyclecomprises: (1) a denaturation step, (2) an annealing step, and (3) apolymerization step. The number of reaction cycles will vary dependingon the application being performed, but will usually be at least 15,more usually at least 20 and may be as high as 60 or higher, where thenumber of different cycles will typically range from about 20 to 40. Formethods where more than about 25, usually more than about 30 cycles areperformed, it may be convenient or desirable to introduce additionalpolymerase into the reaction mixture such that conditions suitable forenzymatic primer extension are maintained.

[0071] The denaturation step comprises heating the reaction mixture toan elevated temperature and maintaining the mixture at the elevatedtemperature for a period of time sufficient for any double stranded orhybridized nucleic acid present in the reaction mixture to dissociate.For denaturation, the temperature of the reaction mixture will usuallybe raised to, and maintained at, a temperature ranging from about 85 to100, usually from about 90 to 98 and more usually from about 93 to 96°C. for a period of time ranging from about 3 to 120 sec, usually fromabout 5 to 30 sec.

[0072] Following denaturation, the reaction mixture will be subjected toconditions sufficient for primer annealing to template DNA present inthe mixture. The temperature to which the reaction mixture is lowered toachieve these conditions will usually be chosen to provide optimalefficiency and specificity, and will generally range from about 50 to75, usually from about 55 to 70 and more usually from about 60 to 68° C.Annealing conditions will be maintained for a period of time rangingfrom about 15 sec to 30 min, usually from about 30 sec to 5 min.

[0073] Following annealing of primer to template DNA or during annealingof primer to template DNA, the reaction mixture will be subjected toconditions sufficient to provide for polymerization of nucleotides tothe primer ends in manner such that the primer is extended in a 5′ to 3′direction using the DNA to which it is hybridized as a template, i.e.,conditions sufficient for enzymatic production of primer extensionproduct. To achieve polymerization conditions, the temperature of thereaction mixture will typically be raised to or maintained at atemperature ranging from about 65 to 75, usually from about 67 to 73° C.and maintained for a period of time ranging from about 15 sec to 20 min,usually from about 30 sec to 5 min.

[0074] The above cycles of denaturation, annealing and polymerizationmay be performed using an automated device, typically known as a thermalcycler. Thermal cyclers that may be employed are described in U.S. Pat.Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610, the disclosures ofwhich are herein incorporated by reference.

[0075] Signal Detection

[0076] The next step in the subject methods is signal detection, i.e.,detecting a change in a fluorescent signal from the FET labeledoligonucleotide probe present in the reaction mixture to obtain an assayresult. In other words, the next step in the subject methods is todetect any modulation in the fluorescent signal generated by the FETlabeled oligonucleotide present in the reaction mixture. The change maybe an increase or decrease in fluorescence, depending on the nature ofthe label employed, but in many embodiments is an increase influorescence. The sample may be screened for an increase in fluorescenceusing any convenient means, e.g., a suitable fluorimeter, such as athermostable-cuvette or plate-reader fluorimeter. Fluorescence issuitably monitored using a known fluorimeter. The signals from thesedevices, for instance in the form of photo-multiplier voltages, are sentto a data processor board and converted into a spectrum associated witheach sample tube. Multiple tubes, for example 96 tubes, can be assessedat the same time. Data may be collected in this way at frequentintervals, for example once every 10 ms, throughout the reaction. Bymonitoring the fluorescence of the reactive molecule from the sampleduring each cycle, the progress of the amplification reaction can bemonitored in various ways. For example, the data provided by meltingpeaks can be analyzed, for example by calculating the area under themelting peaks and this data plotted against the number of cycles.

[0077] The spectra generated in this way can be resolved, for example,using “fits” of pre-selected fluorescent moieties such as dyes, to formpeaks representative of each signaling moiety (i.e. fluorophore). Theareas under the peaks can be determined which represents the intensityvalue for each signal, and if required, expressed as quotients of eachother. The differential of signal intensities and/or ratios will allowchanges in FET to be recorded through the reaction or at differentreaction conditions, such as temperatures. The changes are related tothe binding phenomenon between the oligonucleotide probe and the targetsequence or degradation of the oligonucleotide probe bound to the targetsequence. The integral of the area under the differential peaks willallow intensity values for the FET effects to be calculated.

[0078] Screening the mixture for a change in fluorescence provides oneor more assay results, depending on whether the sample is screened onceat the end of the primer extension reaction, or multiple times, e.g.,after each cycle, of an amplification reaction (e.g., as is done in realtime PCR monitoring).

[0079] Employing Said Assay Result to Determine Whether a PrimerExtension Product is Present in Said Mixture

[0080] The data generated as described above can be interpreted invarious ways. In its simplest form, an increase or decrease influorescence from the sample in the course of or at the end of theamplification reaction is indicative of an increase in the amount of thetarget sequence present, i.e., primer extension product present,suggestive of the fact that the amplification reaction has proceeded andtherefore the target sequence was in fact present in the sample.Quantitation is also possible by monitoring the amplification reactionthroughout the amplification process.

[0081] In this manner, a reaction mixture is readily screened for thepresence of primer extension products. The methods are suitable fordetection of a single primer extension product as well as multiplexanalyses, in which two or more different FET labeled oligonucleotideprobes are employed to screen for two or more different primer extensionproducts. In these latter multiplex situations, the number of differenttypes of probes that may be employed typically ranges from about 2 toabout 20 or higher, usually from about 2 to about 15.

[0082] The above described methods of detecting the presence of one ormore types of primer extension reaction products in a primer extensionreaction mixture finds use in a variety of different applications,representative ones of which are now reviewed in greater detail.

[0083] Utility

[0084] The above described inventive methods find use in a variety ofdifferent applications. In general, the subject oligonucleotide probesand methods of using the same find use in any high fidelity primerextension reaction in which a FET probe and proofreading polymerase areemployed.

[0085] One type of representative application is in monitoring theprogress of nucleic acid amplification reactions, such as polymerasechain reaction applications, including both linear and geometric PCRapplications. As used herein, the term monitoring includes a singleevaluation at the end of a series of reaction cycles as well as multipleevaluations, e.g., after each reaction cycle, such that the methods canbe employed to determine whether a particular amplification reactionseries has resulted in the production of primer extension product, e.g.,a non-real time evaluation, as well as in a real-time evaluation of theprogress of the amplification reaction.

[0086] The subject methods find use in both 5′ nuclease methods ofmonitoring a PCR amplification reaction (e.g., where a Taqman type probeis employed); and non-5′ nuclease methods of monitoring a PCRamplification reaction (e.g., where a molecular beacon type probe isemployed). Again, the subject methods find use in evaluating theprogress of an amplification reaction at a single time (e.g., non-realtime monitoring) and in real-time monitoring.

[0087] Monitoring a PCR reaction according to the subject methods findsuse a variety of specific applications. Representative applications ofinterest include, but are not limited to: (1) detection of allelicpolymorphism; (2) SNP detection; (3) detection of rare mutations; (4)detection of allelic stage of single cells; (5) detection of single orlow copy number DNA analyte molecules in a sample; etc. For example, indetection of allelic polymorphism, a nucleic acid sample to be screened,e.g., a genomic DNA cellular extract, is employed as template nucleicacid in the preparation of a primer extension reaction mixture, asdescribed above, where the reaction mixture includes a different anddistinguishable FET labeled oligonucleotide probe that is specific foreach different allelic sequence to be identified, if present. The assayis then carried out as described above, where the sample is screened fora change in signal from each different oligonucleotide probe. A changein signal from a given probe is indicative of the presence the allelicvariant to which that probe is specific in the sample. Likewise, anabsence of change in signal is indicative of the absence of the allelicvariant in the sample. In this manner, the sample is readily screenedfor the presence of one or more allelic variants. A similar approach canbe used for SNP detection, where a different FET labeled oligonucleotidefor each SNP of interest in a to be screened nucleic acid sample isemployed.

[0088] A significant benefit of employing the subject methods in PCRscreening applications is that the PCR conditions may be “highfidelity,” i.e., they may include a proof reading activity, such thatthe results obtained from the assays performed according to the subjectmethods are highly reliable.

[0089] Kits

[0090] Also provided are kits for practicing the subject methods. Thekits according to the present invention will comprise at least: (a) aFET labeled oligonucleotide, where the kits may included two or moredistinguishable FET labeled oligonucleotides, e.g., that hybridize todifferent target nucleic acids, e.g., two or more different SNPs; and(b) instructions for using the provide FET labeled oligonucleotide(s) ina high fidelity amplification, e.g., PCR, reaction.

[0091] The subject kits may further comprise additional reagents whichare required for or convenient and/or desirable to include in thereaction mixture prepared during the subject methods, where suchreagents include: one or more polymerases, including a polymerase mix,where the one or more polymerases at least include a polymerase thatexhibits proofreading, i.e., 3′→5′ exonuclease activity; an aqueousbuffer medium (either prepared or present in its constituent components,where one or more of the components may be premixed or all of thecomponents may be separate), and the like.

[0092] The various reagent components of the kits may be present inseparate containers, or may all be precombined into a reagent mixturefor combination with template DNA. The subject kits may further comprisea set of instructions for practicing the subject methods.

[0093] In addition to the above components, the subject kits willfurther include instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

[0094] Systems

[0095] Also provided are systems for use in practicing the subjectmethods. The subject systems at least include one or more FET labeledoligonucleotides and a proofreading activity, as well as any otherrequisite components for preparing a primer extension reaction mixture,as described above. In addition, the subject systems may include anyrequired devices for practicing the subject methods, e.g., thermalcyclers, fluorimeters, etc.

[0096] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL PROCEDURES

[0097] General

[0098] The oligonucleotides shown in Table 1 were used for the followingexample. Standard DNA phosphoramidites, including 6-carboxy-fluorescein(6-FAM) phosphoramidite, 5′-Tetrachloro-Fluorescein (TET)phosphoramidite, and 6-carboxytetramethyl-rhodamine (TAMRA) CPG,3′-Dabcyl CPG, were obtained from Glen Research. Black Hole Quenchers(BHQ1, BHQ2, BHQ3) CPG were obtained from Biosearch Technology. EclipseDark Quencher (eDQ) CPG was obtained from Epoch Bioscience. All primerswere purified using Oligo Purification Cartridges (BiosearchTechnology). Doubly labeled FET probes were synthesized using CPGs withvarious quenchers as indicated in Table 1 and with either 6′ FAM-labeledor TET-labeled phosphoramidites at the 5′ end. The doubly labeled FETprobes were purified by preparative HPLC and PAGE using standardprotocols. Phosphorothioate modification was prepared by standardprocedure. TABLE 1 Sequence Listing Name Type Sequence BCL-F1 SEQ ID NO:1 Primer 5′ GGT GGT GGA GGA GCT CTT CAG 3′ BCL-R1 SEQ ID NO: 2 Primer5′ CCA GCC TCC GTT ATC CTG GA 3′ BCL-P1 SEQ ID NO: 3 Probe 5′ FAM-CCTGTG GAT GAC TGA GTA CCT GAA CCG-BHQ1-3′ BCL-P2 SEQ ID NO: 4 Probe5′ FAM-CCT GTG GAT GAC TGA GTA CCT GAA CCG-eDQ-3′ BCL-P3 SEQ ID NO: 5Probe 5′ FAM-CCT GTG OAT GAC TGA GTA CCT GAA CCG-DABCYL-3′ BCL-P4 SEQ IDNO: 6 Probe 5′ FAM-CCT GTG GAT GAC TGA GTA CCT GAA CCG-TAMRA-3′ BCL-P5SEQ ID NO: 7 S-oligo Probe 5′ FAM-CCT GTG GAT GAC TGA GTA CCTGAA*C*C*G-TAMRA-3′ ACT-F1 SEQ ID NO: 8 Primer 5′ GAG CTA CGA GCT GCC TGAC 3′ ACT-R1 SEQ ID NO: 9 Primer 5′ GAC TCC ATG CCC AGG AAG 3′ ACT-P1 SEQID NO: 10 Probe 5′ TET-CAT CAC CAT TGG CAA TGA GCG-BHQ1-3′ ACT-P2 SEQ IDNO: 11 Probe 5′ TET-CAT CAC CAT TGG CAA TGA GCG-eDQ-3′ ACT-P3 SEQ ID NO:12 Probe 5′ TET-CAT CAC CAT TGG CAA TGA GCG-DABCYL-3′ ACT-P4 SEQ ID NO:13 Probe 5′ TET-CAT CAC CAT TGG CAA TGA GCG-TAMRA-3′ ACT-P5 SEQ ID NO:14 S-oligo Probe 5′ TET-CAT CAC CAT TGG CAA TGA*G*C*G-TAMRA-3′ ABCG-R1SEQ ID NO: 15 Primer 5′ CCC AAA AAT TCA TTA TGC TGC AA 3′ ABCG-P1 SEQ IDNO: 16 Primer 5′ FAM-CAG CAT TCC ACG ATA TGG ATT TAC GGC-BHQ1-3′ ABCG-P2SEQ ID NO: 17 Primer 5′ FAM-CAG CAT TCC ACG ATA TGG ATT TAC GGC-TAMRA-3′ABCG-T1 SEQ ID NO: 18 Template 5′ ATC AGC ATT CCA CGA TAT GGA TTT ACGGCA TCA GTT GCA GCA TAA TGA ATT TTT GGG A 3′ MTHFR-F1 SEQ ID NO: 19Primer 5′ GGA AGA ATG TGT CAG CCT CAA AG 3′ MTHFR-R1 SEQ ID NO: 20Primer 5′ CTG ACC TGA AGC ACT TGA AGG AG 3′ MTHFR-P1 SEQ ID NO: 21 WtProbe 5′ TET-TGA AAT CGG CTC CCG CA-BHQ1-3′ MTHFR-P2 SEQ ID NO: 22 MutProbe 5′ FAM-TGA AAT CGA CTC CCG CAG A-BHQ1-3′

EXAMPLE 1 Properties and Use of TET Labeled FET Probes with VariousQuenchers

[0099] Real Time amplifications and detection were performed in ABIPRISM 7700 (Applied Biosystems) using 30 μl reactions that contained 20mM Tris-HCl (pH 8.3), 60 mM KCl, 5.3 mM MgCl₂, 0.2 mM dATP, 0.2 mM dTTP,0.2 mM dGTP, 0.2 mM dCTP, 0.5 μM of each primer, 0.2 μM FET probe,4-fold series dilute of Jurkat cDNAs, either 0.6 units Taq Polymerase(Fisher) or a mix of Taq and Pwo DNA Polymerase (Roche MolecularBiochemicals) at unit ratio of 5 to 1.

[0100] A 99 basepair segment of the human beta actin gene was amplifiedusing primers ACT-F1 and ACT-R1 listed in Table 1. Thermal profile was95° C. 15 sec; 50 cycles of 95° C. 15 sec, 60° C. 30 sec, 72° C. 45 sec.After amplification, fluorescent intensity at each well was measured bypost PCR reading.

[0101] Five types of TET labeled FET probes (ACT-P1, ACT-P2, ACT-P3,ACT-P4 and ACT-P5 as shown in Table 1) were tested in Real Timeamplification. Post PCR reading result is shown in Table 2. The emissionintensity of donor under condition with template is divided by theemission intensity of donor under condition without template to give +/−signal ratio, which indicates weather the probes are degraded or not.For TET labeled FET probes having TAMRA or Dabcyl as quencher, 3′→5′exonuclease addition caused a dramatic decrease in +/− signal ratio dueto cleavage of FET probes. TABLE 2 Fluorescent changes in various TETlabeled FET probe during PCR PCR using Taq pol. PCR using Taq + Pwo pol.donor/quencher no template Template +/− signal ratio no templatetemplate +/− signal ratio TET/BHQ1 4500.0 20250.0 4.5 4500.0 20000.0 4.4TET/eDQ 4000.0 20000.0 5.0 4000.0 20000.0 5.0 TET/Dabcyl 6000.0 19000.03.2 16000.0 24000.0 1.5 TET/TAMRA 3900.0 19000.0 4.9 12000.0 18000.0 1.5TET/TAMRA 4000.0 20000.0 5.0 3900.0 19000.0 4.9 S-oligo

[0102] Real Time amplification plot is shown in FIGS. 1A-E. TET labeledFET probes having either BHQ1 or Eclipse Dark Quencher at 3′-end areresistant to 3′→5′ exonuclease, and are therefore suitable for use inhigh fidelity PCR (FIGS. 1A and 1B). By contrast, TET labeled FET probeshaving either TAMRA or Dabcyl Quencher at 3′-end are degraded by 3′→5′exonuclease, and no proper real time PCR results can be obtained (FIGS.1C and 1D). The TET labeled FET probes having TAMRA Quencher at 3′-endcan also be resistant to 3′→5′ exonuclease if three phosphorothioateinternucleotide linkages at 3′-end are added (FIG. 1E), which confirmsthe reliability of this whole experiment.

EXAMPLE 2 Properties and Use of FAM Labeled FET Probes with VariousQuenchers

[0103] Real Time amplifications and detection were performed in ABIPRISM 7700 using 30 μl reactions that contained 20 mM Tris-HCl (pH 8.3),60 mM KCl, 5.3 mM MgCl₂, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP, 0.2 mMdCTP, 0.5 uM of each primers, 0.2 μM FET probe, various copies of a cDNAclone of human bcl-2 gene or Jurkat cDNAs, 0.6 units Taq DNA Polymerase(Fisher Scientific) or a mix of Taq and Pwo DNA Polymerase (RocheMolecular Biochemicals) at unit ratio of 10 to 1.

[0104] A 190 basepair segment of the human bcl-2 gene was amplifiedusing primers BCL-F1 and BCL-R1 listed in Table 1. Thermal profile was95° C. 15 sec; 50 cycles of 95° C. 15 sec, 60° C. 30 sec, 72° C. 45 sec.After amplification, fluorescent intensity at each well was measured bypost PCR reading.

[0105] Five types of FAM-labeled FET probes (BCL-P1, BCL-P2, BCL-P3,BCL-P4 and BCL-P5 as shown in Table 1) were tested in Real Timeamplification. Post PCR reading result is shown in Table 3. For FAMlabeled FET probes having TAMRA or Dabcyl as quencher, 3′→5′ exonucleaseaddition caused a dramatic decrease in +/− signal ratio due to cleavageof FET probes. TABLE 3 Fluorescent changes in various FAM labeled FETprobe during PCR PCR usin Taq pol. PCR using Taq + Pwo pol.donor/quencher no template template +/− signal ratio no templatetemplate +/− signal ratio FAM/BHQ1 5000.0 20000.0 4.0 5000.0 20000.0 4.0FAM/eDQ 5000.0 20000.0 4.0 5000.0 19000.0 3.8 FAM/Dabcyl 7000.0 20000.02.9 18000.0 25000.0 1.4 FAM/TAMRA 5000.0 22500.0 4.5 20000.0 30000.0 1.5FAM/TAMRA 10000.0 30000.0 3.0 10000.0 29000.0 2.9 S-oligo

[0106] Representative Real Time amplification plot is shown in FIGS.2A-2B. FAM labeled FET probes having either BHQ1 or Eclipse DarkQuencher at 3′-end are resistant to 3′→5′ exonuclease, and are thereforesuitable for use in high fidelity PCR (FIG. 2A). By contrast, FAMlabeled FET probes having either TAMRA or Dabcyl Quencher at 3′-end aredegraded by 3′→5′ exonuclease, and no proper real time PCR results canbe obtained (FIG. 2B). Thus, similar results as Example 1 are repeatedin a different system.

EXAMPLE 3 Properties of FET Oligo as Primers

[0107] PCR amplifications were performed in ABI PRISM 7700 using 30 μlreactions that contained 10 mM Tris-HCl (pH 8.85), 25 mM KCl, 5 mM(NH₄)₂SO₄, 5 mM MgCl₂, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP, 0.2 mMdCTP, 0.5 μM of each primer, 0.2 μM of each primer (ABCG-R1 and ABCG-P1or ABCG-P2), 1 million copies of template (ABCG-T1) or just TE buffer inno template control (NTC), 0.6 units of Pwo DNA Polymerase (RocheMolecular Biochemicals). Thermal profile was 95° C. 10 sec; 40 cycles of95° C. 15 sec, 60° C. 60 sec.

[0108] Two types of FET primers (ABCG-P1 and ABCG-P2) and a template(ABCG-T1) as listed in Table 1 were tested in Real Time Amplification ofhuman ABC transporter ABCG2. Multicomponent analysis is shown in FIG. 3.Thus, the subject FET labeled nucleic acid detectors having a BHQ1 at3′-end are resistant to 3′→5′ exonuclease activity when used as aprimer. By contrast, the FET labeled nucleic acid detectors having aTAMRA at 3′-end are degraded by 3′→5′ exonuclease activity. Similarresults were obtained using FET primers having mismatch at 3′-end.

EXAMPLE 4 Allele Discrimination Under High Fidelity PCR

[0109] An 89 basepair segment of the human methylenetetrahydrofolatereductase (MTHFR) gene was amplified using primers MTHFR-F1 and MTHFR-R1listed in Table 1. Real Time amplifications and detection were performedin ABI PRISM 7700 using 30 μl reactions that contained 20 mM Tris-HCl(pH 8.3), 60 mM KCl, 5.3 mM MgCl₂, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mMdGTP, 0.2 mM dCTP, 0.1 uM of primer MTHFR-F1 and 1 μM of primerMTHFR-R1, 0.2 μM each of FET probes MTHFR-P1 and MTHFR-P2, templatescontaining varies number of MTHFR wild type or mutant or both as listedin table 4, a mix of Taq and Pwo DNA Polymerase (Roche MolecularBiochemicals) at unit ratio of 5 to 1. Thermal profile was 95° C. 15sec; 50 cycles of 95° C. 15 sec, 55° C. 40 sec, 700° C. 40 sec. Afteramplification, fluorescent intensity at each well was measured by postPCR reading and analyzed by allele discrimination software in ABI PRISM7700. Graphical result is shown in FIG. 4 and further summarized inTable 4. TABLE 4 Summary of template types and allele call by PRISM 7700Allele 1 = wild type (TET labeled probe) Allele 2 = mutant type (FAMlabeled probe) Template types Call by Template types Call by Templatetypes Call by Template types Call by (copy num) PRISM 7700 (copy num)PRISM 7700 (copy num) PRISM 7700 (copy num) PRISM 7700 mut: 10⁶ Allele 2mut: 2 × 10⁵ Allele 1/2 mut: 8 × 10³ Allele 1 Wt: 10⁶ Allele 1 wt: 10⁵wt: 10⁵ mut: 10⁶ Allele 2 mut: 2 × 10⁵ Allele 1/2 mut: 8 × 10³ Allele 1Wt: 10⁶ Allele 1 wt: 10⁵ wt: 10⁵ mut: 10⁶ Allele 2 mut: 2 × 10⁵ Allele1/2 mut: 8 × 10³ Allele 1 Wt: 10⁶ Allele 1 wt: 10⁵ wt: 10⁵ mut: 10⁶Allele 2 mut: 2 × 10⁵ Allele 1/2 mut: 8 × 10³ Allele 1 Wt: 10⁶ Allele 1wt: 10⁵ wt: 10⁵ mut: 10⁶ Allele 2 mut: 4 × 10⁴ Allele 1/2 mut: 1.6 × 10³Allele 1 No template No amp wt: 10⁵ wt: 10⁵ wt: 10⁵ Mut: 10⁶ Allele 2mut: 4 × 10⁴ Allele 1/2 mut: 1.6 × 10³ Allele 1 No template No amp wt:10⁵ wt: 10⁵ wt: 10⁵ mut: 10⁶ Allele 2 mut: 4 × 10⁴ Allele 1/2 mut: 1.6 ×10³ Allele 1 No template No amp wt: 10⁵ wt: 10⁵ wt: 10⁵ mut: 10⁶ Allele2 mut: 4 × 10⁴ Allele 1/2 mut: 1.6 × 10³ Allele 1 No template No amp wt:10⁵ wt: 10⁵ wt: 10⁵

[0110] The call by ABI PRISM 7700 matches well with types of templateused (Table 4). No template controls give no amplification signal. Thus,two different allele sequences can be detected and distinguished usingFAM and TET labeled FET probes under high fidelity PCR.

[0111] The above results and discussion demonstrate that FET labelednucleic acid detectors suitable for use in a variety of different highfidelity PCR applications are provide by the subject invention. As such,the subject methods represent a significant contribution to the art.

[0112] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. The citation of anypublication is for its disclosure prior to the filing date and shouldnot be construed as an admission that the present invention is notentitled to antedate such publication by virtue of prior invention.

[0113] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A method for detecting the production of a primerextension product in a primer extension reaction mixture, said methodcomprising: (a) producing a primer extension mixture that includes anucleic acid polymerase having 3′→5′ exonuclease activity and a FETlabeled oligonucleotide that includes a 3′→5′ exonuclease resistantquencher domain; (b) subjecting said primer extension mixture to primerextension reaction conditions; (c) detecting a change in a fluorescentsignal from said FET labeled oligonucleotide probe to obtain an assayresult; and (d) employing said assay result to determine whether aprimer extension product is present in said mixture.
 2. The methodaccording to claim 1, wherein said primer extension reaction is a PCRamplification reaction.
 3. The method according to claim 2, wherein saidmethod is a real-time method of monitoring said PCR amplificationreaction.
 4. The method according to claim 1, wherein said FET labeledoligonucleotide is a nucleic acid detector molecule that includes asingle-stranded target binding sequence linked to fluorophore and darkquencher.
 5. The method according to claim 4, wherein energy transferoccurs between said fluorophore and dark quencher of said FET labeledoligonucleotide probe upon fluorophore excitation when said FET labeledoligonucleotide is not hybridized to target nucleic acid.
 6. The methodaccording to claim 5, wherein energy transfer does not occur betweensaid fluorophore and dark quencher of said FET labeled oligonucleotideprobe upon fluorophore excitation when said FET labeled oligonucleotideprobe is hybridized to a target nucleic acid.
 7. The method according toclaim 3, wherein said method is a 5′ nuclease method of monitoring a PCRamplification reaction.
 8. The method according to claim 7, whereinenergy transfer does not occur between said fluorophore and darkquencher of said FET labeled oligonucleotide probe upon fluorophoreexcitation when said FET labeled oligonucleotide probe is cleaved by 5′nuclease.
 9. The method according to claim 4, wherein said targetbinding sequence comprises a hybridization domain complementary to asequence of said primer extension product.
 10. A method of monitoring ofa PCR amplification reaction, said method comprising: (a) preparing aPCR amplification reaction mixture by combining: (i) a template nucleicacid; (ii) forward and reverse nucleic acid primers; (iii)deoxyribonucleotides; (iv) a nucleic acid polymerase having 3′→5′exonuclease activity; and (v) a FET labeled oligonucleotide thatincludes: a 3′→5′ exonuclease resistant quencher domain comprising adark quencher, a fluorescent reporter domain comprising a fluorophoreand a PCR product complementary domain, wherein fluorescence energytransfer does not occur between said fluorophore and said quencher uponfluorophore excitation when said FET labeled oligonucleotide ishybridized to a product nucleic acid of said PCR reaction; (b)subjecting said PCR amplification reaction mixture to PCR amplificationconditions; (c) monitoring said reaction mixture for a fluorescentsignal from said FET labeled oligonucleotide probe to obtain an assayresult; and (d) employing said assay result to monitor said PCRamplification reaction.
 11. The method according to claim 10, whereinsaid method is a method of monitoring a PCR amplification reaction inreal time.
 12. The method according to claim 10, wherein said FETlabeled oligonucleotide is a probe selected from the group consistingof: scorpion probes, sunrise probes, molecular beacons, andconformationally assisted probes.
 13. The method according to claim 10,wherein said fluorescence energy transfer occurs between saidfluorophore and quencher of said FET labeled oligonucleotide uponfluorophore excitation when said FET labeled oligonucleotide is nothybridized to said product nucleic acid.
 14. The method according toclaim 10, wherein said dark quencher is located at the 3′ end of saidFET labeled oligonucleotide.
 15. The method according to claim 14,wherein said dark quencher has maximum absorbance between about 400 andabout 700 nm.
 16. The method according to claim 15, wherein said darkquencher has maximum absorbance between about 500 and about 600 nm. 17.The method according to claim 16, wherein said dark quencher comprises asubstituted 4-(phenyldiazenyl)phenylamine structure.
 18. The methodaccording to claim 16, wherein said dark quencher has a structurecomprising at least two residues selected from aryl, substituted aryl,heteroaryl, substituted heteroaryl and combination thereof, wherein atleast two of said residues are covalently linked via an exocyclic diazobond.
 19. The method according to claim 18, wherein said dark quenchercomprises a formula:

wherein: R₀,R₁,R₂, R₃, R₄,R₅ are independently: —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, SO₃, —N[(CH₂)_(n)CH₃]₂ wherein n=0to 5 or —CN; R₆ is —H or —(CH₂)_(n)CH₃ where n=0 to 5; and v is a numberfrom 0 to
 10. 20. A method of monitoring of a PCR amplificationreaction, said method comprising: (a) preparing a PCR amplificationreaction mixture by combining: (i) a template nucleic acid; (ii) forwardand reverse nucleic acid primers; (iii) deoxyribonucleotides; (iv) anucleic acid polymerase having 3′→5′ exonuclease and 5′→3′ exonucleaseactivity; and (v) a FET labeled oligonucleotide that includes: a 3′→5′exonuclease resistant quencher domain comprising a dark quencher, afluorescent reporter domain comprising a fluorophore and a PCR productcomplementary domain, where fluorescence energy transfer occurs betweensaid fluorophore and quencher upon fluorophore excitation when said FETlabeled oligonucleotide is not hybridized to said template nucleic acid;(b) subjecting said PCR amplification reaction mixture to PCRamplification conditions; (c) monitoring said reaction mixture for afluorescent signal from said FET labeled oligonucleotide probe to obtainan assay result; and (d) employing said assay result to monitor said PCRamplification reaction.
 21. The method according to claim 20, whereinsaid method is a method of monitoring a PCR amplification reaction inreal-time.
 22. The method according to claim 20, wherein saidfluorescence energy transfer does not occur between said fluorophore andquencher of said FET labeled oligonucleotide upon fluorophore excitationwhen said FET labeled oligonucleotide is hybridized to said productnucleic acid.
 23. The method according to claim 20, wherein saidfluorescence energy transfer does not occur between said fluorophore andquencher of said FET labeled oligonucleotide upon fluorophore excitationwhen said FET labeled oligonucleotide is cleaved by 5′ nuclease.
 24. Themethod according to claim 20, wherein said quencher is located at the 3′end of said FET oligonucleotide.
 25. The method according to claim 24,wherein said dark quencher has maximum absorbance between about 400 andabout 700 nm.
 26. The method according to claim 25, wherein said darkquencher has a maximum absorbance between about 500 and about 600 nm.27. The method according to claim 26, wherein said dark quenchercomprises a substituted 4-(phenyldiazenyl)phenylamine structure.
 28. Themethod according to claim 26, wherein said dark quencher has a structurecomprising at least two residues selected from aryl, substituted aryl,heteroaryl, substituted heteroaryl and combination thereof, wherein atleast two of said residues are covalently linked via an exocyclic diazobond.
 29. The method according to claim 28, wherein said dark quencherhas the following structure:

wherein: R₀,R₁,R₂,R₃,R₄,R₅ are independently: —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, SO₃, —N[(CH₂)_(n)CH₃]₂ wherein n=0to 5 or —CN; R₆ is —H or —(CH₂)_(n)CH₃ where n=0 to 5; and v is a numberfrom 0 to
 10. 30. A method for screening a nucleic acid sample for thepresence of first and second nucleic acids that differ from each otherby a single nucleotide, said method comprising: (a) producing a primerextension mixture that includes: (i) said nucleic acid sample; (ii) anucleic acid polymerase having 3′→5′ exonuclease activity; and (iii)first and second FET labeled oligonucleotide probes that arecomplementary to said first and second nucleic acids, respectively,wherein each of said first and second FET labeled oligonucleotidesincludes a 3′→5′ exonuclease resistant quencher domain; (b) subjectingsaid primer extension mixture to primer extension reaction conditions;(c) detecting a change in a fluorescent signal, if any, from said firstand second FET labeled oligonucleotide probes to obtain an assay result;and (d) employing said assay result to determine the presence or absenceof said first and second nucleic acids in said sample.
 31. The methodaccording to claim 30, wherein said FET labeled oligonucleotide probesare nucleic acid detector molecules that include a single-strandedtarget binding sequence linked to fluorophore and dark quencher.
 32. Themethod according to claim 31, wherein energy transfer occurs betweensaid fluorophore and dark quencher of each of said FET labeledoligonucleotide probes upon fluorophore excitation when said FET labeledoligonucleotide is not hybridized to target nucleic acid.
 33. The methodaccording to claim 31, wherein energy transfer does not occur betweensaid fluorophore and dark quencher of said FET labeled oligonucleotideprobe upon fluorophore excitation when said FET labeled oligonucleotideprobe is hybridized to a target nucleic acid.
 34. The method accordingto claim 31, wherein energy transfer does not occur between saidfluorophore and dark quencher of said FET labeled oligonucleotide probesupon fluorophore excitation when said FET labeled oligonucleotide probeis cleaved by 5′ nuclease.
 35. The method according to claim 30, whereinsaid second nucleic acid is an SNP of said first nucleic acid.
 36. Asystem for use in detecting the production of a primer extension productin a primer extension reaction mixture, said system comprising: (a) aFET labeled oligonucleotide that includes a 3′→5′ exonuclease resistantquencher domain; and (b) a nucleic acid polymerase having 3′→5′exonuclease activity.
 37. The system according to claim 36, wherein saidFET labeled oligonucleotide has said quencher domain located at its 3′end.
 38. The system according to claim 37, wherein said quencher domaincomprises a dark quencher.
 39. The system according to claim 38, whereinsaid dark quencher has maximum absorbance between about 400 and about700 nm.
 40. The system according to claim 39, wherein said dark quencherhas a maximum absorbance between about 500 and 600 nm.
 41. The systemaccording to claim 40, wherein said dark quencher comprises asubstituted 4-(phenyidiazenyl)phenylamine structure.
 42. The systemaccording to claim 40, wherein said dark quencher has a structurecomprising at least two residues selected from aryl, substituted aryl,heteroaryl, substituted heteroaryl and combination thereof, wherein atleast two of said residues are covalently linked via an exocyclic diazobond.
 43. The system according to claim 42, wherein said dark quencherhas the following structure:

wherein: R₀,R₁,R₂,R₃,R₄,R₅ are independently: —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, SO₃, —N[(CH₂)_(n)CH₃]₂ wherein n=0to 5 or —CN; R₆ is —H or —(CH₂)_(n)CH₃ where n=0 to 5; and v is a numberfrom 0 to
 10. 44. A kit for use in detecting the production of a primerextension product in a primer extension reaction mixture, said kitcomprising: (a) a FET labeled oligonucleotide that includes a 3′→5′exonuclease resistant quencher domain; and (b) instructions forpracticing the method according to claim
 1. 45. The kit according toclaim 44, wherein said kit further comprises a nucleic acid polymerasehaving 3′→5′ exonuclease activity.
 46. The kit according to claim 44,wherein said FET labeled oligonucleotide has said quencher domainlocated at its 3′ end.
 47. The kit according to claim 46, wherein saidquencher domain comprises a dark quencher.
 48. The kit according toclaim 47, wherein said dark quencher has maximum absorbance betweenabout 400 and about 700 nm.
 49. The kit according to claim 48, whereinsaid dark quencher has a maximum absorbance between about 500 and 600nm.
 50. The kit according to claim 49, wherein said dark quenchercomprises a substituted 4-(phenyldiazenyl)phenylamine structure.
 51. Thekit according to claim 49, wherein said dark quencher has a structurecomprising at least two residues selected from aryl, substituted aryl,heteroaryl, substituted heteroaryl and combination thereof, wherein atleast two of said residues are covalently linked via an exocyclic diazobond.
 52. The kit according to claim 51, wherein said dark quencher hasthe following structure:

wherein: R₀,R₁,R₂,R₃,R₄,R₅ are independently: —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, SO₃, —N[(CH₂)_(n)CH₃]₂ wherein n=0to 5 or —CN; R₆ is —H or —(CH₂)_(n)CH₃ where n=0 to 5; and v is a numberfrom 0 to
 10. 53. In a method comprising employing fluorescently labelednucleic acid detector and a polymerase having a 3′→5′ exonucleaseactivity, the improvement comprising: employing a FET labeledoligonucleotide that includes a 3′→5′ exonuclease resistant quencherdomain as said fluorescently labeled nucleic acid detector. or employinga FET labeled oligonucleotide that includes a 3′→5′ exonucleaseresistant donor domain as said fluorescently labeled nucleic aciddetector.
 54. The method according to claim 53, wherein said FET labeledoligonucleotide has said quencher domain located at its 3′ end.
 55. Themethod according to claim 54, wherein said quencher domain comprises adark quencher.
 56. The method according to claim 55, wherein said darkquencher has maximum absorbance between about 400 and about 700 nm. 57.The method according to claim 56, wherein said dark quencher has amaximum absorbance between about 500 and 600 nm.
 58. The methodaccording to claim 57, wherein said dark quencher comprises asubstituted 4-(phenyidiazenyl)phenylamine structure.
 59. The methodaccording to claim 57, wherein said dark quencher has a structurecomprising at least two residues selected from aryl, substituted aryl,heteroaryl, substituted heteroaryl and combination thereof, wherein atleast two of said residues are covalently linked via an exocyclic diazobond.
 60. The method according to claim 59, wherein said dark quencherhas the following structure:

wherein: R₀,R₁,R₂,R₃,R₄,R₅ are independently: —H, halogen,—O(CH₂)_(n)CH₃, —(CH₂)_(n)CH₃, —NO₂, SO₃, —N[(CH₂)_(n)CH₃]₂ wherein n=0to 5 or —CN; R₆ is —H or —(CH₂)_(n)CH₃ where n=0 to 5; and v is a numberfrom 0 to 10.